Mouse anti γh2ax

MCF-7 cells were transduced with control and NRMT1 knockdown virus as described above. Cells were treated with vehicle control or 240 μM etoposide. At the indicated time points cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked in 3% bovine serum albumin. γH2AX staining was performed with 1:250 dilution mouse anti-γH2AX (Novus Biologicals, Littleton, CO) followed by Alexa-Fluro 594-conjugated goat anti-mouse secondary at 1:1000 (Invitrogen). NRMT1 staining was performed with 1:200 dilution rabbit anti-NRMT1 [1 (link)] followed by Alexa-Fluro 594 conjugated goat anti-rabbit secondary at 1:1000 (Invitrogen). The cells were counterstained with Hoechst (AnaSpec, Flemont, CA). Cells were visualized using an EVOS FL microscope (Life Technologies). Cells with foci were counted, and the number of foci per cell calculated.

Confocal analyses of LINE-1 ORF1p expression were analysed on a Zeiss LSM 710 confocal microscope using a previously described method [91 (link)]. Antibodies for the immunostaining: Rabbit anti LINE-1 ORF1p, 1:500, a generous gift of Dr. Oliver Weichenrieder (Max Planck, Germany). Secondary antibody: Alexa 488 Donkey anti Rabbit, 1:1,000 (Thermo). Mouse anti γ^+-H2AX, 1:200, clone 3F2 (Novus). Secondary antibody: Alexa 555 Donkey anti Mouse, 1:1,000 (Thermo). Rabbit anti cleaved caspase-3 (cl_Caspase3) (Cell Signalling, 9661S) was used at a 1:200 dilution. Secondary antibody: Alexa Fluor anti Rabbit 555 (1:1,000, Invitrogen). DAPI (Thermo) was used at 1:500. To assess the number of L1-ORF1p cytoplasmic foci in human blastocysts, every second image of a confocal stack was used at the height of the ICM. L1-ORF1p cytoplasmic foci were enhanced by applying a granulation filter with the same parameters to all images, and ORF1p cytoplasmic foci were counted manually. To assess L1-ORF1p⁺- cl_Caspase3⁺ double staining, 6 embryos were stained. To quantify the L1-ORF1p⁺-Cl-csapase3⁺ cells, 4/6 embryos were used.